Robert P. Metzger

Effect of Streptozotocin Diabetes on Acid Phosphatase and Selected Glycosidase Activities of Serum and Various Rat Organs

Summary The activities of the lysosomal hydrolases α-l-fucosidase, α-d-mannosidase, β-d-galactosidase, and N-acetyl-β-d-glucosa-minidase are significantly elevated in epididymal fat tissue and in serum from streptozotocin-diabetic rats compared with those from age-matched normal controls. Acid phosphatase activity is significantly raised over control values in epididymal fat tissue and, perhaps, liver from streptozotocin-diabetic rats. In contrast, rats fasted 72 hr show increased activities of all the glycosidases and acid phosphatase in their liver and serum. Glycosidase and acid phosphatase activities of epididymus, kidney, spleen and testis are not affected by streptozotocin-diabetes. This study was supported in part by U.S. Public Health Service Grant 5685-10.

Protein-bound carbohydrate content of normal and tumorous human lung tissue

Abstract The protein-bound carbohydrate content of normal and tumor tissue (adenocarcinoma, squamous cell carcinoma, and bronchiogenic carcinoma) of the human lung has been determined by gas—liquid chromatography. Cells from three lung tumor types contained from two to four times the amounts of sialic acid, fucose, mannose, galactose. N -acetylglucosamine and N -acetylgalactosamine than tissue taken at a site distant from the tumor. Cells taken from a site adjacent to the tumor had values of protein-bound carbohydrate intermediate between tumor and normal tissue. Approx. 62% of the sialic acid obtained from the tumor sample was neuraminidase labile. These data suggest that human lung tumor cells are synthesizing elevated amounts of glycoprotein which is probably present on the cell surface.

NAD-dependent l-fucose dehydrogenase from sheep liver☆

Abstract l -Fucose dehydrogenase has been purified about 45-fold from the soluble fraction of sheep liver. The enzyme accepts either l -fucose or d -arabinose as substrates but is specific for NAD as coenzyme. Disc-gel clectrophoresis failed to separate the activities obtained with l -fucose and d -arabinose substrates. The pH maximum is 10.4; at this pH, the Km values are 1.5, 7.2, and 0.19 m m for l -fucose, d -arabinose, and NAD, respectively. The product obtained with the l -fucose substrate under these conditions was l -fuconic acid; with a d -arabinose substrate, d -arabonic acid was formed, d -Glucose, d -galactose, d -xylose, and l -arabinose were not substrates for this enzyme.

Stimulation of glucose oxidation by liver homogenates with hexose 6-phosphates.

Abstract Liver homogenates incubated with labeled glucose yield increasing values of 14CO2 as the concentration either of D -glucose 6-phosphate, 2-deoxy- D -glucose 6-phosphate, or D -glucosamine 6-phosphate is increased. The maximal stimulation of 14CO2 production with increasing hexose phosphate concentration comes from systems with added NADP+; added NAD+ does not significantly change values of expired 14CO2 relative to the controls, while added ATP caused dilution in the CO2 labeling. 2-Deoxy-glucose 6-phosphate was the most effective of the hexose phosphates stimulating 14CO2 production, followed by glucosamine 6-phosphate. The former was found to stimulate the production of an unidentified lactone, which, however, was not 6-phosphogluconolactone. 6-Phosphogluconate caused some 14CO2 increase, but this was less than that due to glucose 6-phosphate. None of the non-phosphorylated compounds tested, such as 2-deoxy- D -glucose, causes the stimulation of 14CO2 production, nor does inorganic phosphate or pyrophosphate. D -Gluconate addition results in a slight dilution of the CO2 labeling. Time studies on the rate of 14CO2 production indicate that the phosphate esters themselves are the cause of the increased 14 CO2 values. [I-14C]glucose, when used as substrate, gives about twice the value of expired 14CO2 relative to control as [6-14C]glucose in the presence of increasing concentrations of of glucose 6-phosphate, glucosamine 6-phosphate, or 2-deoxyglucose 6-phosphate. The stimulation of 14CO2 production by 2-deoxyglucose 6-phosphate in the presence of NADP+ was also found using kidney homogenates. Stimulation was not observed using homogenates prepared from heart, spleen or brain. The enzyme, glucose dehydrogenase (β- D -glucose:NAD(P) oxidoreductase, EC i.i.i. 47), has been found in kidney and, previously, in hepatic microsomes. No glucose dehydrogenase activity could be demonstrated in preparations from brain, spleen and heart. The results obtained are the opposite to those predicted on the basis of isotopic dilution by a metabolic intermediate, in addition to those predicted by enzyme inhibition caused by the phosphate esters employed in these experiments. This suggests the involvement of a pathway of glucose oxidation which does not involve glucose 6-phosphate as an intermediate. This pathway, under the conditions employed, is stimulated by NADP+ and certain hexose phosphates and inhibited by ATP.

Solvent affects the conformation of virginiamycin M1(pristinamycin IIA, streptogramin A)

The streptogramins are antibiotics which act by binding two different components at separate nearby sites on the bacterial 50S ribosome, inhibiting protein synthesis. The first component, a macrolactone, is common to many of the streptogramin antibiotics and, thus, is referred to by many names including virginiamycin M1(VM1), pristinamycin IIA, ostreogrycin A and streptogramin A. X-Ray crystallographic studies of VM1 bound to ribosomes and to a deactivating enzyme show a different conformation to that of VM1 in chloroform solution. We now report the results of high resolution 2D NMR experiments that show that the conformation of VM1 in dimethyl sulfoxide and methanol differs from both that in chloroform solution and in the bound form. The 3D structure and the 1H NMR and 13C NMR chemical shifts of VM1 in dimethyl sulfoxide and methanol are described.

A comparison of selected physical properties of hepatic sorbitol dehydrogenases [l-iditol: NAD oxidoreductases] from four mammalian species

1. The sorbitol dehydrogenases [L-iditol: NAD oxidoreductase] from livers of cow, man, rat and sheep each possess molecular weights of about 140,000. The beef, rat and sheep liver enzymes are composed of subunits of molecular weight 40,000. 2. The sorbitol dehydrogenases from livers of these four species each possess an isoelectric point of 7.3. 3. The four enzyme preparations show identical mobilities upon disc-gel electrophoresis and yield a single band of enzymic activity. 4. Sorbitol dehydrogenase activity is activated by the presence of ampholines or by increasing ionic strengths, with maximal activation at about 0.5 M salt concentration. These factors may cause the Km for NAD to be lowered.

Effect of Streptozotocin Diabetes on Selected Enzymatic Activities in Rat Urine

Summary The 24-hr urine collected from streptozotocin-diabetic rats contained activities of acid phosphatase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase, α-L-fu-cosidase, β-D-galactosidase, glutamate-oxal-oacetate transaminase, and α-D-mannosi-dase that were significantly greater than control values. Lactate dehydrogenase activity was similar in urines from diabetic and from control animals. Results were similar with rats injected with streptozotocin from 3 to 30 days prior to use. The levels of urinary acid hydrolase activities present in diabetic rats returned to control values upon treatment with insulin and became elevated above control values once again upon cessation of insulin treatment. The levels of enzymatic activity measured in diabetic rat urine, except for LDH, were directly proportional to the volume of urine produced. However, normal animals with induced polyuria did not produce urines with enzymatic activities above control values. This study was supported by USPHS Grant 5685-12, the California Metabolic Research Foundation, and the Kroc Foundation. M. D. K. was a recipient of a summer fellowship from the California Foundation for Biochemical Research. We thank Dr. Arne N. Wick for his encouragement and for use of his laboratory facilities.

Difference in Conformation of Virginiamycin M1 in Chloroform and Bound Form in the 50S Ribosome or Streptogramin Acetyltransferase

The conformation of virginiamycin M1 (VM1) in chloroform, determined by high-resolution NMR experiments, differs significantly from that of the X-ray crystal structure of VM1 bound to the 50S ribosome and to the active site of a streptogramin acetyltransferase enzyme. This implies that the binding process to these entities causes a major change in VM1 conformation.

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of 14CO2 from L-[1-14C]fucose and D-[1-14C]arabinose been studied in five mammalian species. Cats, guinea pigs, mice, and rabbits respired about 22% of the label of L-[1-14C]fucose or of D-[1-14C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the L-fucose label and 5% of the D-arabinose label in the same time period. Liver homogenates from cat, guinea pig, and rabbit produced significantly more 14CO2 from L-[1-14C]fucose or D-[1-14C]arabinose than mouse or liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low L-fucose dehydrogenase activity. The results suggest that substantial catabolism of L-fucose and D-arabinose occurs in the tissues of some animal species. Investigators wishing to employ L-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize L-fucose to products interfering with their studies.

The effect of fasting, alloxan diabetes, and streptozotocin diabetes on rat liver D-arabinose (L-fucose) dehydrogenase activity.

Abstract The specific activity of d -arabinose ( l -fucose) dehydrogenase is elevated 25, 16, and 20% in the livers of fasted, alloxan diabetic and streptozotocin diabetic rats, respectively, as compared to normal control values. The specific activity of hepatic glucose-6-phosphate dehydrogenase in both groups of diabetic animals was greatly decreased. The same results are obtained if the data are calculated on the basis of activity per gram of wet weight of liver or on the basis of activity per 100 grams body weight. The increase in specific activity of d -arabinose ( l -fucose) dehydrogenase under these conditions may indicate a preferential sparing of the enzyme reflecting a greater utilization of a pathway of l -fucose catabolism.